A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory. This project represents a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Branch (MVDB) and with scientists at the Malaria Research and Training Center (MRTC) at the University of Mali. The hallmark of adaptive immunity is antigen-specific immunological memory. Immunological memory is a phenomenon that having been exposed to a pathogen and survived the infection the experience is remembered by the immune system such that upon re-exposure to the same pathogen an individual's immune response is more rapid and stronger such that the individual may experience no clinical systems of the infection. Indeed, all vaccines are predicated on the phenomenon of immunological memory. However, despite its importance we still have an incomplete understanding of the cellular and molecular mechanisms that underlie the generation, maintenance and activation of immunological memory. Clearly, current efforts to develop a malaria vaccine would benefit from an in-depth understanding of the nature of an effective immune response to the parasite that causes malaria. Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of immunological memory in response to natural malaria infection. In humans, B cell memory is encoded both in long-lived memory B cells and in plasma cells that reside in the bone marrow. The mechanisms by which memory B cells or plasma cells are generated and maintained over a life time are not known. Current evidence, primarily from serological epidemiological studies, indicates that immunological memory to malaria is slow to be acquired, incomplete and short lived. Thus, despite chronic exposure to P. falciparum from birth from infectious mosquito bites, children in endemic areas do not acquire immunity that protects them from severe disease until the age of five. Consequently, children under five years of age are susceptible to severe disease that accounts for over 2 million deaths each year in Africa alone. Acquisition of immunity that protects against severe disease but not against mild disease is acquired prior to adolescence and an immunity sufficient to prevent disease but not to eliminate parasites is acquired only in adolescence to early adulthood. Our current hypothesis is that P. falciparum infection disrupts the normal mechanisms by which memory is generated, maintained or activated. Over the last year we have completed an analysis of the acquisition of malaria-specific MBCs in response to natural infection and have identified an antibody signature associated with malaria immunity. We have also initiated studies to understand the genetic basis of resistance to malaria. To study the acquisition of immunity to malaria we initiated a longitudinal study on a cohort of 225 volunteers, 2-25 years of age in Kambila, a village outside of Bamako, the capital city of Mali in June 2006 prior to the malaria transmission season which runs July through December. This transmission season with six months of malaria exposure and six months free of malaria offers a near ideal condition to evaluate the impact of malaria infection on the generation and maintenance of malaria immunity. Our analysis of the acquisition of malaria-specific MBCs in the Kambila cohort showed that AMA1- and MSP1-specific memory B cells increased with age, likely reflecting a combination maturation of the immune system and continued exposure to malaria. We observed that the number of malaria-specific B cells transiently increased after the first malaria infection. The increase in MBCs was accompanied by a short term rise in the levels of malaria-specific antibody. Of significant interest we also observed a transient increase in the number of MBCs specific for an antigen unrelated to malaria, namely tetanus toxoid (TT). However, the increase in TT-specific MBCs was not accompanied by an increase in the level of TT-specific antibody. Together these novel results suggest that MBCs are maintained in response to nonspecific innate immune system stimulants that accompany infection but the differentiation of MBCs to PCs only occurs in response to antigen. Although we observed what appeared to be an acquisition of typical antigen-specific MBCs in response to malaria infections we also observed by phenotype analysis by flow cytometry a large increase in the number of what have been termed exhausted or atypical MBCs. Atypical MBCs that represent only 1-2% of CD19+ B cells in normal U.S. volunteers represent up to 30% of B cells in Malian children as young as two years of age. We have initiated studies to determine when and how atypical MBCs are generated and their function. Understanding the origin and function of these atypical MBCs should be of significant interest in the development of malaria vaccines. Over the last year we completed studies to assess the nature of the antibody response to P. falciparum in our Kambila cohort in collaboration with Dr. P. Felgner (U.C. Irvine) using a proteome chip containing approximately 25% of the P. falciparum proteome. Our results indicate that the intensity and complexity of the malaria-specific antibody response increased with age and that the intensity and complexity of the serum antibodies at the beginning of the malaria season predicts protection from malaria. We also observed that the complexity and intensity of antibodies transiently rose during the season but most of the increases were lost by six months and did not predict protection. We also identified an antibody signature associated with malaria immunity. A subset of 49 proteins was found to be significantly increased in immune children as compared to nonimmune children. The increased antibody responses specific for the top five of these 49 proteins proved to be a highly sensitive and specific indicator of malaria immunity. Studies are underway to validate the findings in additional sites that differ in the genetic background of the inhabitants and transmission patterns. We have also initiated studies to test the efficacy of these signature proteins as vaccine candidates in animal models of malaria. Lastly, we are developing a microarray containing Var gene products to be used to search for an antibody signature associated with severe malaria.